Doctoral defence: Getnet Balcha Midekessa “Towards understanding the colloidal stability and detection of Extracellular Vesicles”

On 3 March at 13:00 Getnet Balcha Midekessa will defend his doctoral thesis “Towards understanding the colloidal stability and detection of extracellular vesicles”.

Supervisors:
Professor Alireza Fazeli, University of Tartu
Associate Professor Toonika Rinken, University of Tartu

Opponent:
Professor Adriele Prina Mello, Trinity College Dublin (Ireland)

Summary
Nanoparticle (NP) size is an important aspect of cellular uptake by different interactions with biological systems. Aside from their size, NPs also possess surface charge enabling their interaction with their surroundings. EVs are membrane-bound NPs secreted by most cell types under stress. Naturally, EVs exist as colloidal suspensions when resuspended in media and carry a net negative charge due to the presence of lipids, membrane and glycosylated proteins on their surface. It is crucial to maintain EV physical characteristics during storage and surface manipulation, and overcome the issue of EV aggregation for EVs applications. The colloidal stability of EVs is one of the crucial parameters for understanding their fates in different conditions. However, currently there is no a standard methodology to detect and quantify EVs or to assess the colloidal stability of EVs. 

The aim of this study was to determine the colloidal stability of EVs in different conditions and study the applicability of fluorescence NTA (fNTA) for the detection of membranous EVs. The ZP of EVs was influenced by the buffer concentration, detergent, ionic strength and pH. The ZP value of EVs shifts towards less negative values along the increase of buffer concentration. ZP of EVs also shifted to more negative values as pH increased, favoring electrostatic repulsions between EV particles and increasing their colloidal stability. We also assessed the physical characteristics of blood serum EVs derived from healthy and psoriasis patients, but no difference in particle size distribution and ZP values was found. Finally, we showed the effect of membrane dye CellMask™ Green (CMG) labeling on the physical characteristics of JAr EVs isolated using different methods, as well as EVs derived from biological fluids (i.e. bovine follicular fluid and seminal plasma). fNTA analysis revealed that the increase in CMG concentration altered the particle size of fluorescent nanoparticles. Our results also showed the mean particle size of fluorescent and total NPs of JAr EVs were affected by the EV purification method. Biologically derived EVs showed a greater variation than cell culture-derived EVs. Our studies highlight the importance of using standardized ZP measurements and the potential of exploiting fNTA for the detection of membrane dye-labelled EVs.